Phosphoinositides are signaling lipids that are localized to the cytoplasmic surface of cellular membranes. Phosphoinositides regulate vesicle trafficking through unique tagging of membrane compartments and recruitment of effector proteins. PI(3,5)P2 is a minor phosphoinositide in yeast and mammalian cells. PI3P and PI(3,5)P2 function in the endosome/lysosome pathway. Early endosome membranes contain PI3P, while late endsomes/lysosomes contain PI(3,5)P2.

I now report characterization of the pale tremor mouse, a spontaneous mutant characterized by neurodegeneration, diluted pigmentation, and lethality by six weeks of age. Genetic analysis identified a retroviral insertion that generated a null mutation in the gene Fig4, which encodes the 5-phosphatase that dephosphorylates PI(3,5)P2 to PI3P. The level of PI(3,5)P2 is reduced in cultured fibroblasts and there is accumulation of enlarged cytoplasmic vesicles containing the late endosome/lysosome marker LAMP2.

The spontaneous mouse mutant ingls is also characterized by neurodegeneration, diluted pigmentation and juvenile lethality. I identified the causal mutation as the amino acid substitution L156R in Vac14, an activator of the kinase Fab1 that phosphorylates PI3P to generate PI(3,5)P2. ingls fibroblasts also exhibit reduced PI(3,5)P2 and accumulation of enlarged vesicles. The L156R mutation disrupts the interaction of Vac14 and Fab1, localizing the interaction site and demonstrating the importance of Fab1 activation in vivo.

pale tremor mice exhibit peripheral neuropathy and early loss of DRG neurons. I screened 95 patients with Charcot-Marie-Tooth disease (CMT) by exon amplification and sequencing, and identified 4 patients carrying compound heterozygous pathogenic mutations in FIG4. Each patient carried one null allele and the missense mutation, I41T, which exhibits partial function in a yeast assay. To model this disorder, designated CMT4J, a transgenic mouse model carrying the I41T mutation was produced. CMT4J are healthy beyond 7 months of age, demonstrating that the I41T allele retains significant function in vivo.

This thesis demonstrates the significant role of PI(3,5)P2 signaling in mammalian neurons, and expands the range of neurological disease related to phosphoinositide metabolism.